Monosodium glutamate (MSG) is a flavor enhancer commonly used in modern nutrition. In this study, it was aimed to determine the effect of in ovo administered MSG on the embryonic development of thymus, bursa of Fabricius, and percentages of alpha-naphthyl acetate esterase (ANAE) positive lymphocyte by using histological, histometrical, and enzyme histochemical methods in chickens. For this purpose, 410 fertile eggs were used. The eggs were then divided into five groups: group 1 (control group, n = 40 eggs), group 2 (distilled water-injected group, n = 62 eggs), group 3 (0.12 mg/g egg MSG-injected group, n = 80 eggs), group 4 (0.6 mg/g egg MSG-injected group, n = 90 eggs), and group 5 (1.2 mg/g egg MSG-injected group, n = 138 eggs), and injections were performed via the egg yolk. On the 18th and 21st days of the incubation, the eggs were randomly opened from each group until six live embryos were obtained. The embryos of each group were sacrificed by decapitation, and blood, thymus, and bursa of Fabricius tissue samples were taken from the obtained embryos. The MSG-treated groups were found to be retarded embryonic development of thymus and bursa of Fabricius tissue compared to the control and distilled water groups. MSG treatment also resulted in reduced lymphoid follicles count and follicle diameters in bursa of Fabricius (P < 0.05). The percentage of peripheral blood ANAE positive lymphocytes was significantly lower in the MSG-treated groups than in the control and distilled water groups (P < 0.05). In conclusion, it has been found that in ovo administered MSG can adversely affect the embryonic development of thymus and bursa of Fabricius and decrease percentage of ANAE positive lymphocyte.